Böttger Roland, Hoffmann Ralf, Knappe Daniel
Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig, Leipzig, Germany.
Center for Biotechnology and Biomedicine, Universität Leipzig, Leipzig, Germany.
PLoS One. 2017 Jun 2;12(6):e0178943. doi: 10.1371/journal.pone.0178943. eCollection 2017.
Proteolytic degradation of peptide-based drugs is often considered as major weakness limiting systemic therapeutic applications. Therefore, huge efforts are typically devoted to stabilize sequences against proteases present in serum or plasma, obtained as supernatants after complete blood coagulation or centrifugation of blood supplemented with anticoagulants, respectively. Plasma and serum are reproducibly obtained from animals and humans allowing consistent for clinical analyses and research applications. However, the spectrum of active or activated proteases appears to vary depending on the activation of proteases and cofactors during coagulation (serum) or inhibition of such enzymes by anticoagulants (plasma), such as EDTA (metallo- and Ca2+-dependent proteases) and heparin (e.g. thrombin, factor Xa). Here, we studied the presumed effects on peptide degradation by taking blood via cardiac puncture of CD-1 mice using a syringe containing a peptide solution. Due to absence of coagulation activators (e.g. glass surfaces and damaged cells), visible blood clotting was prevented allowing to study peptide degradation for one hour. The remaining peptide was quantified and the degradation products were identified using mass spectrometry. When the degradation rates (half-life times) were compared to serum derived freshly from the same animal and commercial serum and plasma samples, peptides of three different families showed indeed considerably different stabilities. Generally, peptides were faster degraded in serum than in plasma, but surprisingly all peptides were more stable in fresh blood and the order of degradation rates among the peptides varied among the six different incubation experiments. This indicates, that proteolytic degradation of peptide-based therapeutics may often be misleading stimulating efforts to stabilize peptides at degradation sites relevant only in vitro, i.e., for serum or plasma stability assays, but of lower importance in vivo.
基于肽的药物的蛋白水解降解通常被认为是限制其全身治疗应用的主要弱点。因此，人们通常会付出巨大努力来稳定肽序列，使其免受血清或血浆中存在的蛋白酶的影响，血清是全血凝固后获得的上清液，血浆则是添加抗凝剂后离心血液得到的上清液。血浆和血清可以从动物和人类中可重复获得，这使得它们在临床分析和研究应用中具有一致性。然而，活性或活化蛋白酶的谱似乎因凝血过程中蛋白酶和辅因子的激活（血清）或抗凝剂（血浆）对这些酶的抑制而有所不同，例如EDTA（金属和Ca2+依赖性蛋白酶）和肝素（例如凝血酶、因子Xa）。在这里，我们通过使用含有肽溶液的注射器对CD-1小鼠进行心脏穿刺采血，研究了对肽降解的假定影响。由于不存在凝血激活剂（例如玻璃表面和受损细胞），可防止明显的血液凝固，从而能够研究肽在一小时内的降解情况。使用质谱对剩余的肽进行定量，并鉴定降解产物。当将降解速率（半衰期）与从同一动物新鲜获得的血清以及商业血清和血浆样品进行比较时，三个不同家族的肽确实显示出明显不同的稳定性。一般来说，肽在血清中的降解速度比在血浆中快，但令人惊讶的是，所有肽在新鲜血液中更稳定，并且在六个不同的孵育实验中，肽之间的降解速率顺序有所不同。这表明，基于肽的治疗药物的蛋白水解降解可能常常具有误导性，促使人们在仅在体外相关的降解位点（即血清或血浆稳定性测定）稳定肽，而在体内的重要性较低。
